Abstract: Since angiogenesis is one of the hallmarks of cancer, antiangiogenic therapies have been explored as a strategy for cancer therapy. Unfortunately, with current therapies, half of the patients do not respond at all, and only every third patient gains a survival benefit. New insights into the biology of tumor neovasculature are therefore urgently needed to improve antiangiogenic therapy. We apply a multidisciplinary bioengineering approach to explore a new promising target for antiangiogenic therapy. By integrating novel tools for in vitro evaluations and in vivo imaging, we are able to gain unprecedented insight into the biological role of prostate- specific membrane antigen (PSMA) in tumor vessels. The expression of PSMA in tumor neovasculature was already described 19 years ago, yet still little is known about its biological role. We hypothesize that PSMA plays an essential role in angiogenesis of tumor vessels and is therefore a potentially promising therapeutic target. [Our preliminary data has shown that active, angiogenic endothelial cells as well as pericytes expressed high levels of PSMA] and that inhibition of PSMA's enzymatic activity severely impaired the formation of new vessels. Here, we are pairing our new biological insight with innovative technologies to further explore the role of PSMA in tumor neovasculature toward a new antiangiogenetic therapy. To examine the biological role of PSMA in tumor vessels, we will utilize significant bioengineering advancements that will allow us to make direct observations that were previously not possible: (i) A novel cell culturing system for the visualization of PSMA expression over time in EC [and co-cultured other cells such as pericytes; (ii) A prototype microfluidic system to grow a fully vascularized tumor on a chip, allowing us to directly observe the role of PSMA in tumor vascularization; and (iii) A prototype high-resolution optoacoustic scanner to globally interrogate the vasculature and molecular signatures (such as PSMA) in a developing tumor in vivo. Using these tools, developed by a unique consortium of distinctive experts, we will obtain valuable insights into tumor angiogenesis that were not previously possible. Ultimately, we will explore PSMA inhibition as a promising antiangiogenic therapy. We propose to test our hypothesis with three specific aims: In Aim 1, we will explore the role of PSMA in angiogenesis with a new culture method and the microfluidic chip system. We will assess the interplay of PSMA with other markers of angiogenesis and evaluate PSMA inhibition to impair angiogenesis. In Aim 2, we will use the new optoacoustic scanner to explore the role of PSMA in a living, developing tumor. In Aim 3, we will explore the inhibition of PSMA as a novel anti-angiogenetic therapy and monitor tumor development with optoacoustic imaging. Ultimately, this proposal will lead not only to a deeper understanding of PSMA biology but also to a new anti-angiogenetic therapy approach for cancer. [We will also have established novel methods to study tumor vasculature and the tumor microenvironment in a unique way.]